Single‐cell sequencing of mouse heart cellular heterogeneity in hypercholesterolemia reveals the mechanism of myocardial damage

To the Editor, Cardiovascular diseases are a leading cause of death worldwide, accounting for approximately one-third of all deaths.1 As a major risk factor for cardiovascular disease, hypercholesterolemia causes direct myocardial damage. Previous studies have shown that hypercholesterolemia may cause myocardial reactive oxidative stress and mitochondrial dysfunction, ultimately resulting in myocardial damage.2,3 Nevertheless, there is still limited information on the specific molecular mechanisms that underlie this condition. Inflammation is a key process in cardiovascular diseases associated with hypercholesterolemia, such as atherosclerosis, and includes the activation of T lymphocytes.4 Single-cell RNA sequencing is an essential research method for detecting the cellular changes and molecular processes of the heart.5 Currently, single-cell sequencing studies in hypercholesterolemia have mainly focused on the changes in inflammatory cell subtypes in atherosclerotic plaques,6 and the changes in the cellular composition in the heart remain unknown. Therefore, we used single-cell sequencing to detect all heart cells in the early and late stages of disease to explore the molecular mechanism of myocardial damage. First, we utilized Apoe−/− mice fed a high-cholesterol diet to establish a hypercholesterolemia model. In the process, the mouse serum lipoprotein index of TC and LDL increased significantly, and HE and ORO staining indicated that lipid levels increased (Figure S1A–E). Then, we identified 12 cell clusters of heart tissues according to the marker genes via single-cell sequencing (Figure 1A–D). When ordering these clusters according to abundance, the most abundant cell population was fibroblasts. Inflammatory cells, such as myeloid cells, B cells and T cells, increased in the disease group (Figure 1E). Next, cardiomyocytes were identified based on the cardiomyocyte marker genes Ttn and Tnnt2 (Figure 1D).


Single-cell sequencing of mouse heart cellular heterogeneity in hypercholesterolemia reveals the mechanism of myocardial damage
To the Editor, Cardiovascular diseases are a leading cause of death worldwide, accounting for approximately one-third of all deaths. 1 As a major risk factor for cardiovascular disease, hypercholesterolemia causes direct myocardial damage. Previous studies have shown that hypercholesterolemia may cause myocardial reactive oxidative stress and mitochondrial dysfunction, ultimately resulting in myocardial damage. 2,3 Nevertheless, there is still limited information on the specific molecular mechanisms that underlie this condition. Inflammation is a key process in cardiovascular diseases associated with hypercholesterolemia, such as atherosclerosis, and includes the activation of T lymphocytes. 4 Single-cell RNA sequencing is an essential research method for detecting the cellular changes and molecular processes of the heart. 5 Currently, single-cell sequencing studies in hypercholesterolemia have mainly focused on the changes in inflammatory cell subtypes in atherosclerotic plaques, 6 and the changes in the cellular composition in the heart remain unknown. Therefore, we used single-cell sequencing to detect all heart cells in the early and late stages of disease to explore the molecular mechanism of myocardial damage.
First, we utilized Apoe −/− mice fed a high-cholesterol diet to establish a hypercholesterolemia model. In the process, the mouse serum lipoprotein index of TC and LDL increased significantly, and HE and ORO staining indicated that lipid levels increased (Figure S1A-E). Then, we identified 12 cell clusters of heart tissues according to the marker genes via single-cell sequencing ( Figure 1A-D). When ordering these clusters according to abundance, the most abundant cell population was fibroblasts. Inflammatory cells, such as myeloid cells, B cells and T cells, increased in the disease group ( Figure 1E).
Next, cardiomyocytes were identified based on the cardiomyocyte marker genes Ttn and Tnnt2 ( Figure 1D).
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Cardiomyocytes highly expressed the atria-specific genes Sln, Nppa, My17 and Myh6, suggesting that they were associated with atrial function 7 (Table S4). Additionally, cardiomyocytes were mainly distributed in disease samples ( Figure 1E). Therefore, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to further investigate cardiomyocyte function. Interestingly, there was significant enrichment of genes associated with diabetic cardiomyopathy (DCM) in cardiomyocytes, so we selected the cardiomyocyte-specific related gene Myl7 and the DCM-related gene Pln to verify the changes in these cell clusters by immunofluorescence colocation analysis (Figures 1F,G and S1F). The results suggested that Myl7 and Pln expression and the number of cardiomyocyte clusters were increased, and the Pln protein levels were also significantly increased ( Figure 1H,I).
Then, we mapped the immune microenvironment in the heart under hypercholesterolemia. T cells demonstrated significant changes in the differentially expressed gene analysis, so they were further subdivided into six subpopulations. The changes in the abundance of proliferating T cells and Tn cells showed the opposite trend, which might indicate an activated differentiation process (Figure 2A-E). Trajectory analysis showed that naive cells differentiated into two major branches, Th cells and proliferating populations ( Figures 2F and S2A). The proportion of Th cells and CD8+ T cells increased significantly during treatment with a high-cholesterol diet; this was especially true for Th cells ( Figure 2C). Immunofluorescence staining for the specific markers T and Th cells, CD3 and CD4, also showed that Th cells were significantly infiltrated in the disease group ( Figure 2H). 8 The Il-17a, Ccr6 and Cd163l1 genes were highly expressed, illustrating that the Th-cell subpopulation was Th17 cells ( Figure 2G). Immunohistochemical results revealed obvious positivity for IL-17, revealing that Th17 cells mainly accumulated in Additionally, IL-17a is an important cytokine secreted by Th17 cells, and the ELISA and WB results from tissue homogenates showed that IL-17a and IL-17-related proteins were significantly increased in our study ( Figure 2I-K).
Additionally, fibroblasts were further subdivided into seven subpopulations ( Figure 3A,B). The proportion of Cluster 2 was increased as the disease ( Figure C, Table S10). According to the gene ontology (GO)/KEGG analysis of Cluster 2, inflammatory genes and the IL-17 signalling pathway were obviously enriched ( Figure 3D,E). Previous studies suggested that the IL-17 signalling pathway is related to myocardial fibrosis. 9 Our results revealed that fibrosis was more serious in the disease groups, as shown in Figure 3G. We confirmed the source of IL-17 via immunofluorescence staining. The results showed that the majority of IL-17-positive cells were vimentin+ fibroblasts, except in rare cases where α-actinin + cardiomyocytes and rarely costained with CD31. These findings suggest that IL-17 mainly originates from fibroblasts, which is consistent with a prior study ( Figure 3G). 10 The genes and pathways that were enriched in fibroblast Cluster 3 were associated with collagen deposition (Figure 3H,I). Sirius staining confirmed this increase in collagen content. Type I and III collagen fibres were obviously deposited, and the collagen III protein expression level in fluorescence localization was higher in the 8/16-week HCD groups ( Figure 3J,K). Moreover, GO/KEGG analysis of Cluster 6 suggested that fibrosis-related genes and the Wnt pathway were enriched, and the levels of wnt5a and β-catenin proteins, which are key mediators of the Wnt-signalling pathway, were significantly increased in cardiac tissues (Figures 3F,L and S1G,H).  Next, we further performed clustering analysis for macrophages. Macrophages are a subtype of myeloid cells, and they accounted for the largest proportion of this cell type ( Figure 4A-C). Macrophages were subdivided into six subpopulations via marker genes. Among these subpopulations, Cluster 1 was the most abundant, and Cluster 2 showed an increasing trend ( Figure 4D-F). Moreover, the enrichment analysis of Cluster 1 showed that leukocyte migration and chemotaxis were obviously enriched, and the function of Cluster 2 was mainly related to the apoptotic pathway (Figures 4G and S2B). Macrophages influence T-cell activation by regulating MHC-II and may affect fibroblasts through TGF-β pathways ( Figures 4H,I  and S2E,F). In CellChat analysis, T cells, fibroblasts and macrophages had significant and obvious interactions (Table S8), and CXCL12-CXCR4 and CCL5-CCR5 were significant chemokine interactions among them ( Figure 4J,K). Additionally, the smooth muscle cell subpopulation Cluster 5 also expressed the fibroblast markers Dcn and Lum and was thus determined to be myofibroblasts (Table S17). Lymphatic endothelial cells, plasma cells and activated B cells were mainly distributed in disease samples, which may indicate the activation of inflammatory responses ( Figure S3A-F).
In summary, hypercholesterolemia is critical in cardiovascular diseases, and there is an urgent need to develop effective therapeutics. The high expression of Pln genes in cardiomyocytes may provide insight into the possible causes of myocardial injury. The interaction among macrophages, T cells, fibroblasts and collagen deposition emphasize the importance of fibrosis and inflammatory activation. This interaction might partly explain the mechanisms of hypercholesterolemia and provide treatment strategies for cardiovascular diseases.